Reflectance spectra and some respiratory reactions of bovine, equine and human thrombocytes.

GOUCHER, CHARLES R. AND W. KOCHOLATY. (Army Med.ResearchLab., Fort Knox, Ky.) Rejectance spectra and some respiratory reactions of bovine, equine and human thrombocytes. Am. J. Physiol. 188(2) : ~p5-q~~ =957*-Reflectance spectra of human, bovine and equine thrombocytes reveal the existence of pigments which absorb in the visible region of the spectrum. The chromatic properties of these pigments change with the redox potential of the cell. These spectra alterations suggest the existence of a cytochrome system, but the position of the absorption bands does not permit their identification with known mammalian cytochromes. However, platelet homogenates contain a cytochrome oxidase which oxidizes mammalian cytochrome c and which is inhibited by sodium azide. Platelet extracts also contain a DPNH oxidase system which is inhibited by sodium azide. BSERVATIONS of biochemical processes 0 occurring in the living, intact cell form not only an essential complement in the reconstruction of biochemical reactions occurring in solution or suspension, but furnish highly credible evidence of the nature of the contribution of an isolated chemical event to the physiology of the cell. The recent development of sensitive spectrophotometric equipment of high resolution (I, 2) has made possible studies of respiratory enzyme function in vivo. The present communication concerns observations in the respiratory enzyme system of intact mammalian thrombocytes using the integrating sphere in conjunction with a sensitive spectrophotometer (3). The activity of a mammalian cytochrome c oxidase and a DPNH oxidase system has been demonstrated in platelet extracts. MATERIALS AND METHODS Preparation of Platelet Suspensions. Bovine platelets. Blood was collected in the abatoir with acidcitrate-dextrose (ACD)] as an anticoagulant from the slashed neck veins of cattle in siliconed containers and cooled immediately in ice. All subsequent operations were carried out at a temperature of approximately 4OC. Platelet-rich plasma was obtained by passage of blood Received for publication September IO, 1956. l 2.45 gm dextrose, 0.8 gm citric acid, 2.2 gm trisodium citrate/roe ml, 150 ml of such a solution were used for every liter of blood collected. through the Cohn Blood Fractionator at 3600 rpm using siliconized equipment and plastic tubing throughout. Further separation of the platelets from the other formed elements was accomplished in a refrigerated International centrifuge by repeated recentrifugation and redistribution of the platelet-rich layer in cold saline (4, 5). From I gallon of bovine blood was obtained &--254 ml of packed platelets, essentially free from erythrocytes and leucocytes. Such suspensions in saline were used immediately following preparation for the determination of reflectance spectra. Epine p2ateZets. Blood was collected from several horses by venipuncture of the carotid or jugular vein with ACD as an anticoagulant and cooled immediately. After passage through the Cohn Blood Fractionator the obtained platelet-rich plasma was further freed from erythrocytes and leucocytes by fractional centrifugation and washing with cold saline and maintained at 4OC. From I gallon of blood about 3-4 ml of packed platelets was obtained. Human platelets. Human blood was collected by phlebotomy using identical concentrations of ACD anticoagulant as above and platelet-rich plasma was obtained by passage of the blood through the Cohn Blood Fractionator at 3600 rpm. From four donors of the same blood group a total of about 2-3 ml of packed and washed platelets was obtained from 4 pints of blood comparatively free from the other formed elements of blood. Reflectance Measurements. Reflectance spectra of the platelet suspensions were obtained using the integrating sphere (6) with the Beckman Recording Spectrophotometer (7) against an asbestos standard. The platelet suspensions were contained in a zo-mm diameter quartz cuvette (8) having a 3.a-mm light path and a volume of 2.2 ml. The procedures used to determine the reflectance 415 by 10.0.33.2 on A uust 3, 2017 http://ajple.physiology.org/ D ow nladed fom 416 CHARLES R. GOUCHER AND W. KOCHOLATY spectra of the platelets of the three species were as follows: following the separation from the white and red corpuscles the platelets were washed 3 times in o.85y0 cold saline and the suspensions were brought to concentrations suitable for spectrophotometric analysis. Curve I in each of figures I, 2 and 3 was obtained from platelets in the absence of added substrate which were bubbled with a slow stream of oxygen to avoid excessive foaming. Curve z in each case was obtained after addition of potassium ferricyanide to the platelet suspension. A subsequent addition of sodium hydrosulfite resulted in the reflectance spectra represented in each case by czcrve 3. Determination of Cytochrome c Oxidase. A commercial preparation of cytochrome c (Sigma) from horse heart was used for all oxidase determinations. To IO ml of 0.1 M, PH 7.0, potassium phosphate buffer 0.0117 gm of cytochrome c was added. This quantity of cytochrome was reduced by a pinch of Na&Ob. After a s-minute incubation with hydrosulfite a vigorous stream of O2 was passed through the solution to remove the residual reducing agent. 1.0 ml of this cytochrome c solution was used per 2.0 ml of reactants in each cytochrome determination. Following the suggestion of Paul (9) the absorption spectrum of cytochrome c was determined from 400 to 600 rnp at intervals of I rnp. This spectrum revealed that the major P-peak of the cytochrome used had a maximum at 548 rnp. After hydrosulfite treatment the percentage of reduced cytochrome present was estimated using the convention of Smith (IO) ; however, the ratio OD548/ OD565 was used in place of the ratio ODg5o/ODs6~. With hydrosulfite reduction ODs48/OD565 was IO. Essentially identical results were obtained with Palladium-Ha reduction of cytochrome c, while the OD ratio using 550 and 548 rnp differed only by 0.1 of an optical density unit.